Instrument for measuring blood clotting times

ABSTRACT

An improved method and apparatus for monitoring and studying blood clotting times are disclosed. The method includes the step of passing a direct current through a blood or plasma sample and observing the electrical resistance of the sample during the clotting process. The electrical resistance, when plotted against time, is found to form a sigmoid curve having distinct inflections which signify the occurrence of both gellation and fibrin cross-linking within the clotting sample. The apparatus of the present invention includes a sample container which is preferably a disposable cup having a pair of electrodes suitably mounted in it and connected to an electrical connector. A current source is provided for supplying a constant current to the plasma or blood sample, and is equipped with an electrical coupling for mating with the connector on the disposable sample container. A chart recorder is coupled to the constant current source for providing a continuous indication of the sample resistance.

United States Patent [191 Stoner et al.

INSTRUMENT FOR MEASURING BLOOD CLOTTING TIMES Filed:

Inventors: Glenn E. Stoner, 108 David Terrace; Thompson H. Boyd, III, 1620 Jefferson Park Ave., Apt. 12B, both of Charlottesville, Va. 22903 Aug. 20, 1973 Appl. No.: 389,672

US. Cl. 324/65 R, 73/64.1, 128/2 G,

Int. Cl GOlr 27/02 Field of Search 324/65 R, 65 P, 30 B; 128/2 G, 2.1 R, 2.1 E, 2 E; 73/15 A, 64.1;

References Cited UNITED STATES'PATENTS Page 128/2 G Young 128/2 G Paulson et al 324/30 B X Maltby 324/61 R 51 Oct. 8, 1974 Primary Examiner-Stanley T. Krawczewicz Attorney, Agent, or FirmOblon, Fisher, Spivak, McClelland & Maier [57] ABSTRACT An improved method and apparatus for monitoring and studying blood clotting times are disclosed. The method includes the step of passing a direct current through a blood or plasma sample and observing the electrical resistance of the sample during the clotting process. The electrical resistance, when plotted against time, is found to form a sigmoid curve having distinct inflections which signify the occurrence of both gellation and fibrin cross-linking within the clotting sample. The apparatus of the present invention includes a sample container which is preferably a disposable cup having a pair of electrodes suitably mounted in it and connected to an electrical connector. A current source is provided for supplying a constant current to the plasma or blood sample, and is equipped with an electrical coupling for mating with the connector on the disposable sample container. A chart recorder is coupled to the constant current source for providing a continuous indication of the sample resistance.

20 Claims, 12 Drawing Figures PAIENIEI; SET 81574 SIIEET 1 IF 3 WHEATSTONE BRIDGE ELECTROMETER CHART RECORDER 5.64% ozqwmu TIME IMIN.)

COMPLETELY DIGESTED DIGESTED FIRM FIRM

AFTER l2 HOURS APPRECIABLY LYSED APPRECIABLY APPRECIABLY use!) RETRACTED RETRACTED I FIGBA TIME (MIN) INSTRUMENT FOR MEASURING BLOOD CLOTTING TIMES BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates generally to a method and apparatus for measuring blood clotting times, and more particularly, to a method and apparatus for measuring the direct current resistance of blood, and thereby analyzing the blood clotting process.

2. Description of the Prior Art Blood clotting tests are among the most common analyses now performed in hospital laboratories, since blood clotting times are an important parameter in treating patients suffering from numerous types of thromboembolic disorders. However, inthe past, most efforts in the study of blood clotting have been directed toward a gross determination of coagulation times determined by one or more obvious physical characteristics of clotting blood. Nevertheless, it is known that the clotting of blood comprises several significant stages including the initial formation of a gel of linear strands of fibrin, followed at a later time by a cross-linking of the linear strands to form a stable, insoluble clot.

Most measurements of blood clotting times are directed only toward determining the time at which the initial gel of linear fibrin strands is formed, and do not indicate or in any way detect the subsequent crosslinking of the strands, which causes the ultimate stabilization of the blood clot. However, recent scientific discoveries have indicated that the cross-linking step is extremely important in detecting several pathological conditions. In particular, some peoples blood lacks the capability to undergo the cross-linking step naturally, while in other instances, it has been found that certain drugs impede the cross-linking process. For example, most drugs now under study for their cancer inhibiting effects appear to inhibit the cross-linking process. It also appears from scientific research that tumor metastasis has a defnite connection with the cross-linking function. It is therefore clear that information pertaining to the cross-linking capability of a patients blood would be valuable to a physician in many clinical situa tions as well as being an important parameter in hematology research.

Previously known techniques for measuring blood clotting times have included several crude mechanical techniques, including tilting a sample and observing its viscosity or inserting a tool into a sample to test the changing viscosity of the sample. Optical techniques have also been used in which photocells are employed to measure the increasing opacity or turbidity of a clotting plasma sample. Such optical measurements are limited in their practicality, however, by the need to use normally transparent plasma, rather than whole blood, and by the fact that the opacity or turbidity of the clotting sample increases very quickly at the time of gellation, and does not permit subsequent measure ments of the cross-linking phenomenon.

More recently electrical impedance measuring tech niques have been applied to the study of blood clotting phenomena. In particular, reference is directed to U.S. Pat. No. 3,699,437 to Amiram Ur issued Oct. 17, 197.2. The device disclosed in this patent employs 'a test cell containing blood to be examined, and a control cell, containing an additional sample of the blood being 2 studied, but including an anti-coagulating ingredient. The test cell and the control cell form two arms of a Wheatstone bridge, the remaining two arms of which are made up of balancing impedances. A high frequency current on the order of 10 kHz is then used to drive the Wheatstone bridge arrangement containing the blood samples to be tested. The bridge is initially balanced, or maintained unbalanced by a fixed amount, so that its output provides a relative measurement of the impedance ofthe blood in the test cell with respect to that of the blood in the control cell. By continuously adjusting the balance of the bridge, an impedance curve is obtained which has a minimum that appears to coincide with blood coagulation. Accordingly, the apparatus of the Ur patent appears only to provide an electrical measurement of the point of blood coagulation. The apparatus required is, however, very complicated and sensitive in operation. More particularly, the bridge arrangement requiring both a test and a control cell is inherently complex and requires a duplication of parts in that a pair of identical blood sample cells are required. Perhapsmore important is the fact that the apparatus disclosed in the Ur patent requires the use of a high frequency alternating current. The use of a high frequency alternating current naturally results in an impedance which has both reactive and resistive components. Accordingly, in order to balance the bridge both delicate capacitive and delicate resistive adjustments must be made. The delicate nature of such adjustments clearly raises a strong possibility of error in any measurements made. Furthermore, if the bridge arrangement is to be made automatically balancing, complicated circuits are required to permit such automatic balancing to be carried out accurately. In addition to these problems, the need for generating a stable high frequency current provides further possibilities of error and creates the need for additional expensive and complicated electrical equipment. Finally, even if the apparatus disclosed in the Ur patent is constructed and operates perfectly, the output information it produces is simply a measure of the time of initial blood coagulation, which is substantially the same information as was available in the past using other, simpler techniques of measurement. No information is provided regarding the cross-linking process noted above.

Accordingly, a need exists for an improved apparatus for analyzing blood clotting which provides information concerning not only the time of initial coagulation, but also the time at which the cross-linking process takes effect. A need also exists for an apparatus which can provide this information and which is nevertheless inexpensive to produce, simple to operate and accomodates inexpensive and disposable sample containers in conformity with modern hospital needs.

SUMMARY OF THE INVENTION Accordingly, one object of this invention is to provide a novel electrical apparatus for studying the blood clotting process.

Another object of this invention is the provision of a novel, low cost, highly accurate apparatus for analyzing the blood clotting process.

Yet another object of this invention is the provision of a novelapparatus for providing a measure of the time at which the cross-linking process begins during blood clot formation.

A still further object of this invention is the provision of a novel method for analyzing the blood clotting process utilizing a constant direct current.

Another object of this invention is the provision of a unique method for studying the cross-linking process in blood clotting which includes the step of measuring changes in the direct current resistance of a plasma or whole blood sample.

A still further object of this invention is the provision of a novel apparatus including a disposable sample container for use in blood coagulation analysis.

Briefly, these and other objects of the invention are achieved by providing a single sample cell containing a small quantity of blood to be studied. A constant current source is utilized to pass a constant direct current through the blood sample contained in the sample cell. The voltage across the cell and the resulting electrical resistance are monitored in order to study the blood clotting process. The resistance of the sample is found to increase substantially at the time of initial coagulation, and is found to again increase substantially at a later time when the cross-linking process takes place.

BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

FIG. 1 is a block diagram of one form of the apparatus of the present invention used in combination with a conventional photocell arrangement;

FIG. 2 is an illustration of one form of sample container which can be used with the apparatus of the present invention;

FIG. 3A is a graphical representation of the optical density of a sample versus time;

FIG. 3B is a graphical representation of the electrical resistance of a sample versus time;

FIG. 4 is a table illustrating the solubility of plasma clots in MCA and in SK;

FIG. 5A is a graphical representation of the optical density of a sample versus time;

FIG. 5B is a graphical representation of the resistance of a sample versus time for a number of different sample mixtures;

FIG. 5C is a table defining the various curves illustrated in FIG. 5B;

FIG. 6 is a graphical representation of voltage versus time for positive and negative electrode potentials;

FIG. 7 is an illustration of a disposable sample container for use with the apparatus of the present invention;

FIG. 8 is an illustration of the apparatus of the present invention utilizing a disposable container of the type illustrated in FIG. 7; and

FIG. 9 is a block diagram illustrating the electrical components of the apparatus of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Before describing in detail the method and apparatus of the present invention, a brief summary of the blood clotting process is believed to be in order. It is well known that fibrinogen, the clotting protein in plasma, has a molecular weight of about 340,000 and is comprised of three pairs of peptide chains, a(A), B(B) and y, which are held together by disulfide bridges. Prior to fibrin formation, thrombin cleaves a portion of the a(A) and [3(8) peptide chains from the N-terminal end of the fibrinogen molecule, between the arginine and glycine residues.

Blombackand Vestermark have shown that peptide A, the fragment cleaved from the a(A) chain, is released with fibrin formation, and that peptide B, the fragment cleaved from the [3(8) chain is releaed after an appreciable amount of fibrin has been formed (Isolation of Fibrionopeptides by Chromotography", Arkiv Kemi, 12, 173, 1958). The remaining portion of the fibrinogen molecules aggregate, and in the presence of factor XIII, thrombin and Ca ions form a cross-linked gel which is insoluble in solvents such as one per cent monochloracetic acid and 5M urea.

In the initial stages of fibrin formation, fibrin monomers come together forming a noncross-linked gel. After a time lag which is proportional to the fibrinogen/factor XIII concentration ratio, there is a rapid followed by a slower formation of insoluble crosslinked fibrin.

The present invention permits further understanding and monitoring of these later steps in fibrinogen polymerization in situ.

Referring now to the drawings, wherein like reference numerals designate identical or corresponding parts throughout the several views, and more particularly FIG. 1 thereof, an apparatus is illustrated in block diagram from which was used in carrying out the initial research in accordance with the present invention. The apparatus includes an incubation cell 10 in which a sample container 12 is positioned. A white light 14 is also positioned within the incubation cell 10 for projecting a light beam through the sample container 12 to a photocell 16. The output of the photocell 16 is fed to a Wheatstone bridge 18, the output of which is, in turn, fed to a chart recorder 20. In the embodiment illustrated in FIG. 1, the chart recorder is preferably a dual channel Hewlett Packard Model 7100 BM recorder, although any equivalent recorder may be used.

A pair of electrodes 22 are mounted within the sample container 12, and are coupled to an electrometer 24. The electrometer 24 is preferably a conventional solid-state instrument similar to a voltmeter but having an extremely high input resistance (approximately 10 megohms, for example). The electrometer supplies a small direct current to the electrodes 22 and measures the voltage across the electrodes, whereby the resistance of the sample material can be obtained. The output of the electrometer is also coupled to the chart recorder 20 so that one channel of the cart recorder graphs the output of the Wheatstone bridge 18 while the other channel of the recorder simultaneously graphs the output of the electrometer 24.

Referring now the FIG. 2, the sample container 12 is shown in greater detail as including a glass or plastic envelope 26 having an open upper end which is covered by a plastic cap 28. The envelope 26 is shown as having a diameter of 12mm to indicate the general size and the volume range thereof. Naturally, envelopes of other diameters could also be used. A quantity of plasma 30 fills the lower portion of the envelope 26 and provides the sample fluid of which the clotting times are to be measured. Naturally, whole blood could be used in place of the plasma 30, although plasma was more suitable for the initial experiments which used a photocell arrangement to verify the resistance readings in view of the fact that plasma is normally transparent while whole blood is not.

The electrodes 22 are rigidly mounted in plastic cap 28 so that they will be properly positioned within the envelope 26 when cap 28 is in place. Each of the electrodes 22 includes a spherical gold microelectrode 32 which is coupled to the electrometer through a fine wire 34 formed of a highly conductive material. The gold microelectrodes 32 each have an area of approximately 4 sq. mm and are preferably formed from melting and shaping a 2.5 cm length of 0.008 in. gold wire. A pair of plain glass or plastic capillary tubes 36 are mounted in the plastic cap 28 for supporting the gold micro-electrodes 32 in their proper position, and also for insulating the portion of the wires 34 which extend into the envelope 26. Naturally, other types of insulation could also be used, but it is preferable that the insulation material be relatively rigid in nature to maintain an accurate spacing between the gold microelectrodes 32. The spacing between the two electrodes is preferably in the range of from 1.50mm to 2.0mm, although it may be slightly beyond either extremity of this range. The diameter of the gold micro-electrodes is preferably within the range of from 0.97mm to l.l0mm, although, again, this range may be extended somewhat.

When the wires 34 are coupled to the electrometer 24 (which may be a Kiethley Model 600A electrometer, for example) and a suitable plasma sample 30 is positioned within the envelope 26, experimental measurements are begun. An initial test of the method of the present invention was carried out by obtaining platelet poor plasma by collecting whole blood from a fasting donor into standard donor bags anti-coagulated with citrate phosphate dextrose. The whole blood was centrifuged at 5500 rpm at 4 C for five minutes to remove the red blood cells. The remaining plasma was then centrifuged at the same speed and temperature for 60 minutes to remove most of the platelets. The platelet poor plasma was stored in 2 ml.aliquots at 25C. The plasma was polymerized by thawing for 9 minutes at 37 C, glass activating for 3 minutes, and recalcifying with 0.2 ml of 0.25 M CaCl The plasma in a 12 by 75 mm glass tube was then placed in the incubation cell in the position illustrated in FIG. 1. The photocell arrangement was used in conjunction with the electrometer to relate changes in opacity and/or turbidity to the changes in electrical resistance of the sample.

After the plasma sample was recalcified, as described above, the plasma remained in the liquid phase for several minutes. Then, as the plasma started to gel, it became more optically dense, so that less light was transmitted through it to the photocell 16. After gellation, there was no further recorded change in the opacity of the clot. The recalcification time t for the glass activated plasma is approximately 3% minutes, as is shown by a curve 38 in FIG. 3A. The curve 38, which is produced by the chart recorder measuring the output of Wheatstone bridge 18 is a graphical indicating of the opacity of the plasma sample as a function of time. The curve 38 begins at an initial point 40 which indicates a minimum opacity while the plasma is still in its liquid phase. The opacity indicated by the curve remains approximately constant for an interval of about two minutes and then begins to increase rapidly, reaching the recalcification time 2% at approximately 3% minutes, then continues to increase rapidly until approximately 6 minutes have elapsed, at which time the curve 38 flatens out at a maximum level of opacity.

Attention is now directed to FIG. 3B which illustrates the electrical resistance as a function of time of the same sample for which the opacity is illustrated in FIG. 3A; Curve 42 of FIG. 3B was measured using the apparatus illustrated in FIGS. 1 and 2 with the electrometer 24 driving a constant direct current of 0.8 microamp through the plasma sample. As shown in FIG. 3B, the direct current resistance of the plasma sample displays a plurality of very clear inflections. As is apparent from FIG. 3B the resistance curve 42 initially increases to a plateau 44 within only one-half minute after the testing procedure is begun. The electrical resistance of the plasma then increases markedly at time A, producing a clear inflection in the curve 42. After a short time lag, the electrical resistance again shows another sharp increase between points B and C, producing a second slightly broader inflection in the curve 42. The curve subsequently levels off to a nearly constant resistance after a relatively long interval in the region of the point D. The point A represents a time immediately after gellation, while B represents a time just prior to the second inflection of theelectrical resistance curve. The point C is a point just after the second inflection in the resistance curve and the point D represents a time interval of 20 minuts from the initiation of the experiment.

In order to determine the physical significance of the various inflections in the resistance curve, samples of the plasma were removed for analysis at the times represented by the points A, B, C and D. A 2 percent solution of monochloracetic acid (MCA) was then added to each of the samples to terminate further polymerization, and the sample clots were incubated at 37 C in separate solutions of 1 percent monochloracetic acid and streptakinase (SK, 5,000 units/ml). After 12 hours in 1% monochloroacetic acid, the plasma clots in which polymerization was terminated at times A and B were appreciably lysed, while those clots polymerized to time C and D remained retracted. The results of these tests are summarized in the table shown in FIG. 4. Similarly, plasma clots polymerized to time A were completely digested after 12 hours in streptakinase. The clots polymerized to time B were appreciably digested in streptakinase whereas clots polymerized to times C and D remained firm. The contrasting soluability of the plasma clots indicated that very few intercovalent bonds had formed in clots taken at times A and B, while clots taken at times C and D had appreciably more covalent cross-linking bonds, making them insoluble in the monochloroacetic acid. Similarly, clots taken at times A and B were more susceptible to enzymatic breakdown by a fibrinolytic agent than clots taken at times C and D. Again, these results are summarized in the table of FIG- 4.

In addition to the tests mentioned above, the plasma was also recalcified in the presence of a cross-linking inhibitor, glycine methyl ester. Such an inhibitor does not effect the initial gellation of plasma, but greatly reduces covalent bondformation between fibrin molecules by competing for glutaminyl acceptor sites along the fibrin molecules. The results of these tests using the cross-linking inhibitor are illustrated in FIGS. 5A, 5B

and C. FIG. 5A includes a curve 46 which again illustrates increasing opacity versus time of the plasma sample, in substantially the same manner as curve 38 of FIG. 3A. FIG. 5A is merely provided as a reference for comparison with FIG. 5B which includes a plurality of resistance curves for samples of plasma containing different amounts of glycine methyl ester. FIG. 5C is a table relating specific concentrations of glycine methyl ester to the various curves of FIG. 5B. In FIG. 5B curve 48, drawn as a solid line, represents a concentration of only 25 mM of glycine methyl ester in the plasma sample. The curve inflections are quite similar in their timed relationship to the inflections illustrated in curve 42 of FIG. 3B. However, by gradually increasing the concentration of glycine methyl ester to 65 mM (curve 50) and to a concentration of 70 mM of glycine methyl ester (curve 52), the second inflection point is gradually moved to the right, indicating that the inhibiting ingredient caused the cross-linking process to occur at a substantially later time. Finally, at a concentration of 75 mM glycine methyl ester, as illustrated by curve 54, the second inflection in the resistance curve was completely eliminated.

These studies indicate that the second inflection noted in the resistance curve was due to the crosslinking phenomenon and more particularly, the the polymerization of fibrinogen molecules. Accordingly the present invention, in providing an indication of the time at which fibrinogen polymerization takes place, provides information which was not hitherto available from conventional testing, whether mechanical, optical or electrical.

Studies concerning what occurred at the surface of the micro-electrodes 32 have also been conducted. By measuring the electrical potential of each of the gold micro-electrodes with a calomel reference electrode, it was found that the electrical potential of the positive electrode increases twice during plasma polymerization, as shown by a curve 56 in FIG. 6. This increase in potential at the positive electrode is attributable to the absorption of a negatively charged species, possibly the negatively charged fibrinopeptides, which uncover cross-linking sites as they are released during fibrin formation. The potential of the negative electrode, illustrated by a curve 58 fluctuates only slightly as is shown by curve 52 in FIG. 6.

Referring now to FIG. 7, a second embodiment of a sample container is illustrated. The sample container 60 is designed to be disposable, and is adapted for rapid attachment to, and detachment from, a suitable power source and measuring apparatus. The sample container 60 includes a plastic cup body 62 which may be made of any conventional plastic material, such as polyethylene. The upper open end of the plastic cup body 62 is preferably formed into a rim 64 which is adapted to frictionally engage a removable plastic cap 66. A pair of electrodes 68 is positioned within the plastic cup body 62 for contacting a sample 70 of plasma or whole blood within the sample container 60. The electrodes 68 are preferably constructed in the same manner as the electrodes 22 discussed earlier, and include spherical gold micro-electrodes connected to fine wires which are insulated by relatively rigid capillary tubes or other suitable insulating material. The spacing between the electrodes 68 is preferably approximately 2.0mm, although it may be within the range noted hereinabove. The electrodes 68 are mounted in a reinforced base member 72 which is either adhered to a lower surface of the plastic cup 72, or formed integral therewith. The contacts 68 are also electrically coupled to a pair of male plugs 74 which are rigidly mounted in the lower outwardly facing surface of the reinforced base 72. The male plugs 74 may be of any variety suitable for coupling the electrodes 68 to a power source and measuring apparatus of the type illustrated in FIG. 8.

FIG. 8 is an illustration of an apparatus 76 which includes the components illustrated in block diagram form in FIG. 9. The apparatus 76 includes a sample receptical and plug 78 mounted on its upper surface for physically receiving the sample container and for providing an electrical connection with the plugs 74 coupled to the electrodes 68. The apparatus 76 includes a plurality of controls 80 for permitting its operation and also includes a plurality of indicators 82 for monitoring the status of operation of the apparatus 76. The output of the apparatus may be in the form of a chart 84, for example, on which are indicated the resis tance measurements obtained from the blood sample contained in the sample container 60.

The components of the apparatus 76 illustrated in FIG. 8 are shown in block diagram form in FIG. 9. In general, the apparatus 76 accomplishes the combined functions of the electrometer 24 and chart recorder 20, illustrated in FIG. 1. However, for practical use of the present invention, it is preferable to construct a single purpose apparatus including a chart recorder or equivalent output means and a constant current source and high impedance voltmeter combination designed exclusively for the purpose of blood clot testing. Accordingly, as shown in FIG. 9, the apparatus 76 includes a constant direct current source 86 for providing a constant current of approximately 0.8 microamp to a sample 88, as described above. More particularly, the sample 88 is preferably as illustrated in FIG. 7, wherein the constant current from the source 86 is applied through the plugs 74 and the electrodes 68 to the blood sample 70. A high impedance voltmeter 90 is coupled across the sample 88 to measure the voltage across the sample, so that the resistance of the sample may be obtained. The output of the high impedance voltmeter may be fed directly to a chart recorder 92, for producing curves of the type illustrated in FIG. 3B, or alternatively, a differentiator 94 may be positioned between the output of the voltmeter and the input of the chart recorder 92. The differentiator 94 would produce spiked outputs upon the occurrence of the inflections noted in the resistance curve, to more clearly delineate the times at which the inflections occur. Furthermore, suitable timing circuits 96 may be coupled to the chart recorder 90 to place timing marks on the output chart 84. The timing marks are preferably set to occur such that they define the time at which gellation and crosslinking occur in normal blood. Thus, the distance between the timing marks and the marks generated by the sample 88 through the differentiator 94 provide an indication of the behavior of the blood sample being tested relative to a normal or standard which is arbitrarily established. Accordingly, by simply reviewing the relationship of the marks on the output chart 84, a physician can easily determine whether a blood sample is normal, and whether the gellation or cross-linking phenomena are occurring in accordance with a normal time schedule. This information can be very important in diagnosing the illnesses noted above, and also in indicating whether certain medications should be prescribed.

It is again noted that the present invention relies upon the use of a direct current in measuring blood impedance. The direct current passing through a fluid such as blood produces a potential known as the Ada potential which is analagous to a PN junction potential, between the electrons which are the charge carriers in the measuring electrodes and the ions which are charge carriers in the blood itself. This potential becomes a portion of the output measurement when direct current is used, as in the present invention, while it is not present in the output of devices which use alternating current, such as conventional impedance measuring devices of the type described in the Ur patent.

The apparatus illustrated in FIGS. 7 9 can be produced relatively inexpensively by the use of a single level constant current and by the use of a specialized chart recorder. The chart recorder can also be replaced by other types of indicating devices, such as digital readouts, meters, warning lights, buzzers, etc., where desired.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein. v

Accordingly, what is claimed as new and desired to be secured by Letters Patent of the United States is:

1. A method of measuring the times of formation of fibrin and the cross-linking of fibrin in whole blood comprising the steps:

placing a pair of electrodes into a blood sample;

supplying a direct current to said electrodes;

measuring the electrical impedance of said sample between said electrodes; and

detecting inflections in said electrical impedance which occur during said step of measuring, said inflections denoting the occurrence of fibrin formation and cross-linking of said fibrin.

2. A method as in claim 1, further comprising the step of:

maintaining said direct tude.

3. A method as in claim 2, wherein said step of maintaining includes the step of:

fixing said direct current at an amplitude ofapproximately 0.8 microamp.

4. A method as in claim 1, further comprising the step of:

recording said inflections of said electrical impedance after said step of measuring.

5. A method as in claim I further comprising the step of:

timing the occurrence of said inflections.

6. A method as in claim ll, further comprising the step of:

differentiating said inflections to produce clearer indications of the occurrence of said inflections.

7. A method as in claim ll, wherein said step of measuring includes the step of:

continuously monitoring the direct current electrical resistance of said sample.

8. A method as in claim 4, further comprising the step of:

current at a constant amplirecording timing marks representing standard times i for fibrin formation and cross-linking in juxtaposition with said inflections to permit comparison of the behavior of said sample with a reference standard. V

9. A method as in claim 1, wherein said step of placing includes the steps of:

obtaining a sample of whole blood,

placing said wholeblood sample into a sample container having a pair of micro-electrodes mounted therein; and

coupling said micro-electrodes to a source of direct current.

10. An apparatus for detecting changes in the impedance of a biological fluid comprising:

sample container means for holding a sample of said biological fluid,

electrode means positioned within said sample container means for continuously engaging said sample of biological fluid,

power source means coupled to said electrode means for passing a direct current through said sample,

measuring means coupled across said electrode means for measuring changes in the resistance of said sample; and

indicator means coupled to said measuring means for indicating the output of said measuring means whereby changes occurring in the molecular structure of said biological fluid are detected through indicationsof said changes in resistance.

11. An apparatus as in claim 10, further comprising:

differentiatingmeans coupled between said measuring means and said indicator means for emphasizing changes in the output of said measuring means.

12. An apparatus as in claim 10, further comprising:

timing means coupled to said indicator means for providing a standard for comparison with the output of said measuring means.

13. An apparatus as in claim 10, wherein:

said sample container means comprises a disposable vessel formed of a plastic material and said electrode means are mounted within said vessel.

14. An apparatus as in claim 13, wherein said sample container means further comprises:

an electrical connector mounted to a bottom portion of said vessel and electrically connected to said electrode means for connecting said electrode means to said power source.

15. An apparatus as in claim 10, wherein said electrode means comprises:

a pair of electrodes, each including a spherical gold micro-electrode coupled to a fine conductive wire, saidfine wire surrounded by a firm tube of insulating material.

16. An apparatus as in claim 10, wherein saidpower source means comprises:

an electric circuit for generating a fixed amplitude direct current.

17. An apparatus as in claim 10, wherein said measuring means comprises:

a very high impedance voltmeter.

18. An apparatus as in claim 10, wherein said indicator means comprises:

said vessel; and

an electrical connector mounted in said reinforced base member for coupling said electrodes to a power source.

20. A disposable sample container as in claim 19,

wherein each of said electrodes comprises:

a spherical gold micro-electrode coupled to a fine conductive wire, said fine wire surrounded by a firm tube of insulating material.

- UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No 3,8+O,806 Dated October a, 197

- Inventor(s) Glenn E. S'tOnI et a1.

It is eertified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1, after the Title and before "BACKGROUND OF THE INVENTION? insert the following paragraph:

- The invention described herein was made in the course of work under a grant or award from the Department of Health, Education and Welfare and therefore maybe manufactured and used by or for the Government for-governmental purposes without the payment of any royalties thereon or therefore.

- Signed and sealed this 17th day of December 1974.

(SEA-L) Attest:

. licCOY If. GIN-102'? JR. I a i C. 1-'IARSHAI..I.. DAMN 1 Atte'sting Officer v Commissioner-of Patents FORM PC4050 (10-69) USCOMM-DC 60376-P69 u.s. sovzmmzm PRINTING OFFICE: 930 

1. A method of measuring the times of formation of fibrin and the cross-linking of fibrin in whole blood comprising the steps: placing a pair of electrodes into a blood sample; supplying a direct current to said electrodes; measuring the electrical impedance of said sample between said electrodes; and detecting inflections in said electrical impedance which occur during said step of measuring, said inflections denoting the occurrence of fibrin formation and cross-linking of said fibrin.
 2. A method as in claim 1, further comprising the step of: maintaining said direct current at a constant amplitude.
 3. A method as in claim 2, wherein said step of maintaining includes the step of: fixing said direct current at an amplitude of approximately 0.8 microamp.
 4. A method as in claim 1, further comprising the step of: recording said inflections of said electrical impedance after said step of measuring.
 5. A method as in claim 1 further comprising the step of: timing the occurrence of said inflections.
 6. A method as in claim 1, further comprising the step of: differentiating said inflections to produce clearer indications of the occurrence of said inflections.
 7. A method as in claim 1, wherein said step of measuring includes the step of: continuously monitoring the direct current electrical resistance of said sample.
 8. A method as in claim 4, further comprising the step of: recording timing marks representing standard times for fibrin formation and cross-linking in juxtaposition with said inflections to permit comparison of the behavior of said sample with a reference standard.
 9. A method as in claim 1, wherein said step of placing includes the steps of: obtaining a sample of whole blood, placing said whole blood sample into a sample container having a pair of micro-electrodes mounted therein; and coupling said micro-electrodes to a source of direct current.
 10. An apparatus for detecting changes in the impedance of a biological fluid comprising: sample container means for holding a sample of said biological fluid, electrode means positioned within said sample container means for continuously engaging said sample of biological fluid, power source means coupled to said electrode means for passing a direct current through said sample, measuring means coupled across said electrode means for measuring changes in the resistance of said sample; and indicator means coupled to said measuring means for indicating the output of said measuring means whereby changes occurring in the molecular structure of said biological fluid are detected through indications of said changes in resistance.
 11. An apparatus as in claim 10, further comprising: differentiating means coupled between said measuring means and said indicator means for emphasizing changes in the output of said measuring means.
 12. An apparatus as in claim 10, further comprising: timing means coupled to said indicator means for providing a standard for comparison with the output of said measuring means.
 13. An apparatus as in claim 10, wherein: said sample container means comprises a disposable vessel formed of a plastic material and said electrode means are mounted within said vessel.
 14. An apparatus as in claim 13, wherein said sample container means further comprises: an electrical connector mounted to a bottom portion of said vessel and electrically connected to said electrode means for connecting said electrode means to said power source.
 15. An apparatus as in claim 10, wherein said electrode means comprises: a pair of electrodes, each including a spherical gold micro-electrode coupled to a fine conductive wire, said fine wire surrounded by a firm tube of insulating material.
 16. An apparatus as in claim 10, wherein said power source means comprises: an electric circuit for generating a fixed amplitude direct current.
 17. An apparatus as in claim 10, wherein said measuring means comprises: a very high impedance voltmeter.
 18. An apparatus as in claim 10, wherein said indicator means comprises: a chart recorder for continuously recording the output of said measuring means.
 19. A disposable sample container for use with an apparatus for detecting changes in the impedance of a biological fluid comprising: a cup-shaped vessel formed of a plastic material, a reinforced base member secured to a bottom portion of said vessel, a pair of electrodes mounted in said reinforced base member and protruding into the interior volume of said vessel; and an electrical connector mounted in said reinforced base member for coupling said electrodes to a power source.
 20. A disposable sample container as in claim 19, wherein each of said electrodes comprises: a spherical gold micro-electrode coupled to a fine conductive wire, said fine wire surrounded by a firm tube of insulating material. 